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Among these harvested plants, the plants t … Among these harvested plants, the plants that had fresh weight over 13 mg were used for GC-TOF/MS analysis (WT, n = 17; mto1, n = 13; and tt4, n = 20). For liquid chromatography-quadrupole-time-of-flight/mass spectrometry (LC-Q-TOF/MS) analysis, 3 biological replicates were prepared. All the plant materials were frozen immediately in liquid nitrogen to quench the enzymatic activity.<br /><br />
Extraction and sample preparation for GC-TOF/MS analysis<br />
Each sample was extracted with a concentration of 25 mg fresh weight of tissues per μl extraction medium (methanol/chloroform/water [3:1:1 v/v/v]) containing 10 stable isotope reference compounds using a Retsch mixer mill MM310 at a frequency of 30 Hz for 3 min at 4°C. Each isotope was adjusted to a final concentration of 22.5 ng per μl injection [49,50]. After centrifugation for 5 min at 15,100 × g, 400 μl of the supernatant was used for further analysis. The extracts were evaporated to dryness in a Savant SPD2010 SpeedVac Concentrator (Thermo Electron Corporation, Waltham, MA, USA).<br />
For methoximation, 20 μl of methoxyamine hydrochloride (20 mg/ml in pyridine) was added to the sample. After 30 h of derivatization at room temperature, the sample was trimethylsilylated for 1 h using 20 μl of MSTFA with 1% TMCS at 37°C with shaking. Twenty μl of n-heptane was added following silylation. All the derivatization steps were performed in the vacuum glove box VSC-100 (Sanplatec, Japan) filled with 99.9995% (G3 grade) of dry nitrogen. The analysis of metabolites by GC-TOF/MS was performed as described previously.<br /><br /> cribed previously.<br /><br />
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